1. Field of the Invention
The present invention relates generally to the field of virology and to the process and control of viral RNA transcription. More specifically, the present invention relates to a Cys3-His1 motif within the M2-1 protein of respiratory syncytial virus and the use of this functional motif as a target for screening of antiviral agents.
2. Description of the Related Art
Human respiratory syncytial (RS) virus is a member of the pneumovirus genus of the Paramyxoviridae. It is the leading viral cause of pediatric lower respiratory tract disease and is a significant cause of morbidity and mortality worldwide. The genome of RS virus is a single strand of negative-sense RNA 15,222 nucleotides in length having 10 genes encoding 11 proteins (4, 15, 25, 33). As with all nonsegmented, negative-sense RNA viruses, RNA synthesis requires a genomic RNA encapsidated with nucleocapsid (N) protein and the virus encoded components of the RNA dependent RNA polymerase, the phosphoprotein (P) and the large polymerase protein (L) (7, 38). The N, P and L proteins are sufficient for replication of the genomic RNA (10, 38). However, RS virus, unlike other nonsegmented negative-sense RNA viruses, encodes an additional protein, M2-1, which functions during transcription of the viral mRNAs (3). This protein has been shown to increase the processivity of the viral polymerase thus preventing premature termination during transcription. Additionally it has been shown that the M2-1 protein enhances readthrough of transcription termination signals and thus functions as a transcription antiterminator (8, 13). Furthermore, the RS virus gene end sequences vary and it has been shown that the M2-1 protein acts differentially at the different gene ends (11).
The M2-1 protein is found only in pneumoviruses. It is encoded by the first of two open reading frames (ORF) in the next to last gene of the RS virus genome and is 194 amino acids in length (calculated m.w.=22,150 daltons) (5). M2-1 is a hydrophilic protein with a predicted pI of 9.6. Examination of the predicted amino acid sequence led to the identification of a Cys3-His1 motif (Cxe2x80x94X7xe2x80x94Cxe2x80x94X5xe2x80x94Cxe2x80x94X3xe2x80x94H) located near the amino terminus of the protein from residues 7 to 25. This motif is found in the M2-1 protein of all the pneumoviruses examined to date (12, 20, 37, 39). A similar motif is found in VP30 of the filoviruses (29). A number of motifs have been characterized as coordinating the binding of zinc. These motifs have been grouped according to the arrangement and number of cysteine and histidine residues involved in coordinating the zinc ion. Many proteins bind zinc, and, in a number of enzymes, zinc has been shown to play a role in catalysis (16). However, in other cases, zinc plays a purely structural role (24). The Cys3-His1 motif has been characterized in only one protein, Nup475, a mammalian transcription factor in which it was demonstrated to bind zinc and a structure for this motif has been proposed using NMR and photometric analyses (36).
Six species of the RS virus M2-1 protein have been observed by two-dimensional electrophoresis (27). When the M2-1 protein was analyzed by SDS-PAGE under reducing conditions, the majority of the protein was found in two forms distinguished by their electrophoretic mobility. The cause of the differences in migration of these species or whether the different species have different functions is unknown. The M2-1 protein has been reported to be phosphorylated, but the relationship between phosphorylation and the different forms of the protein has not been investigated (18).
The M2-1 protein has also been shown to interact with the N protein in RS virus-infected cells or when the two proteins are coexpressed in cells from plasmid vectors (9, 28). The significance of this interaction and the role it may play is currently unknown.
The prior art is deficient in methods of screening for antiviral compounds that are specific for respiratory syncytial virus. The present invention fulfills this long-standing need and desire in the art.
In the instant invention the role of the predicted zinc coordinating residues of the Cys3-His1 motif in the antitermination activity of M2-1 protein, in its ability to interact with N protein, and in its phosphorylation state were examined. It was found that mutations of the residues predicted to coordinate zinc prevented the M2-1 protein from enhancing transcriptional readthrough and interacting with the nucleocapsid protein. It was also found that the two major species of the M2-1 protein distinguished by their mobility in reducing SDS-PAGE differed according to whether they were phosphorylated. This work demonstrates the requirement for conservation of the Cys3-His1 motif, a potential zinc binding domain, to maintain the functional integrity of the M2-1 protein.
One object of the present invention is to provide a method of screening for antiviral compounds specific for respiratory syncytial virus.
In one embodiment of the present invention, there is provided a method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: a) treating a sample of respiratory syncytial virus with a compound, thereby producing a treated sample and an untreated sample; b) producing respiratory syncytial virus RNA transcripts in the presence of said treated sample or in the presence of said untreated sample; and c) comparing said transcripts produced in the presence of said treated sample with said transcripts produced in the presence of said untreated sample, wherein less readthrough transcripts due to termination at gene end signal produced in the presence of said treated sample is indicative of an antiviral compound directed towards respiratory syncytial virus.
In another embodiment of the present invention, there is provided a method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: a) treating a sample of respiratory syncytial virus with a compound, thereby producing a treated sample and an untreated sample; and b) comparing the treated sample with an untreated sample, wherein an inhibitory effect on the treated sample compared to the untreated sample of a characteristic such as M2-1 transcriptional antitermination, zinc binding, phosphorylation, binding to respiratory syncytial virus N protein, viral transcription or generation of progeny virus particles is indicative of a compound with antiviral activity.
In another embodiment of the present invention, there is provided a method of screening for antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: a) treating a sample of respiratory syncytial virus with a chelator or a compound that inhibits binding of Zinc; and b) comparing the treated sample with an untreated sample, wherein an inhibitory effect on the treated sample compared to the untreated sample of a characteristic such as M2-1 transcriptional antitermination, zinc binding, phosphorylation, binding to respiratory syncytial virus N protein, viral transcription or generation of progeny virus particles is indicative of a compound with antiviral activity.
In yet another embodiment of the present invention, there is provided a method of screening antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: a) treating a sample of respiratory syncytial virus selected from the group consisting of core polymerase protein, nucleocapsid protein, phosphoprotein, an isolated virus or a virus-infected cell with a compound, thereby producing a treated sample and an untreated sample; and b) producing respiratory syncytial virus RNA transcripts in the presence of said treated sample or in the presence of said untreated sample, wherein an inhibition of virus RNA transcription or production of progeny virus particles in the presence of said treated sample is indicative of an antiviral compound directed towards respiratory syncytial virus.
In yet another embodiment of the present invention, there is provided a method of designing antiviral compounds directed towards respiratory syncytial virus, comprising the steps of: a) designing a compound that inhibits zinc binding to a Cys3-His1 motif of a respiratory syncytial virus M2-1 protein; b) treating a sample of respiratory syncytial virus with said designed compound; and c) comparing said treated sample with said untreated sample, wherein an inhibitory effect on said treated sample, when compared to the untreated sample, of a characteristic such as M2-1 transcriptional antitermination, zinc binding, phosphorylation, binding to respiratory syncytial virus N protein, viral transcription or generation of progeny virus particles is indicative of a compound with antiviral activity. A preferred method of designing is by computer modeling.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.